Effect Of α-Zearalanol On Osteoblasts Cultured In Vitro

Objects:To investigate the effect of Effect ofα-ZAL on osteoblasts cultured in vitro. It studied the effect ofα-ZAL on the proliferation and differentiation and protein expression of osteoblast in rats,in order to approach the mechanism of action of isoflavones phytoestrogen in bone metabolism.The further objective is to develop medicine of preventing and curing osteoporosis.Methods:1 Osteoblasts were obtained from the calvaria of newborn SD rats with enzymatic digestion method.2 Application of calcium cobalt method of alkaline phosphatase (ALP) staining and mineralized nodules staining to identify osteoblast.3 Inspect the effect ofα-ZAL on the proliferation of osteoblast by MTT methods.4 Detect the change of the activity of ALP in osteoblast in 24h and 48h usingρ-nitrophenylphosphate(PNPP)disodium matrix dynamics methods.5 Detect the expressions of Bcl-2 and Bax protein using immuno-chemical staining.Result:1 Cell morphology observation: The original osteoblasts were first seeded were spherical, After cultured for 24 hours, the living cells would adherent; the second day, the cells would expand and become triangular , spindle-shape, the nucleus were significantly increased. In the five day, the cells could be seen diversity, the cytoplasm was abundant and stretched out, the surrounding cells would become semi-fusion by pseudopodias. Culturing for one week, the cells became long, polygonal, cord-shaped; when the cells were fusion would become cobblestone-like.2 The identification of osteoblasts: From the ALP staining, we could see the black particles or massive sink in the cytoplasm of 90% cells, so the cells are osteoblasts. From the mineralized nodules staining, we could see the Orange nodules and the boundary was definition.3 The effect on the proliferation of osteoblast: After cultivated 24h in the concentrations ofα-ZAL (10^-5mol/L¹0^-10 mol/L)and (10^-10mol/L )E2 group, blank control group, we could see that Compared with E₂ group,α-ZAL was at the two concentrations of 10^-7mol / L, 10-10mol / L was not significant difference (P> 0.05), that means the two concentrations ofα-ZAL could promote osteoblast proliferation.4 The influence ofα-ZAL on osteoblast's ALP:We could see that three groups were significantly different with the normal group, the E2 group,α-ZAL was at the two concentrations of 10-7mol / L, 10-10mol / L was not significant difference (P> 0.05). It means that the two concentrations ofα-ZAL can promote the cell secreted ALP. As increasing the number of days with medication, the ALP levels also increased.5 The influence ofα-ZAL on Bcl-2,Bax protein expression:α-ZAL (10^-7mol / L ) group,α-ZAL (10^-10mol / L) group could inhibit osteoblast's apoptosis.Conclusion:1 Certain dose ofα-ZAL can promote the proliferation and differentiation of osteoblast.This consequence illustrates that they has activity on bone just like estrogen.2 Bax/Bcl-2 involved in apoptosis of osteoblasts and a certain concentration ofαzeranol can play a key regulatory role by Bax/ Bcl-2.